1. Field of the Invention
The present invention provides a novel bone-associated protein. More particularly, the protein of the present invention is named OSF-6, and belongs to a group of transcription control factor molecules. In addition, this OSF-6 can be obtained from bone of mammals, including mice and humans. Moreover, the present invention provides a process for producing OSF-6 by recombinant gene technology using cultured cells such as animal cells.
The diseases generally referred to as bone metabolic diseases may include osteoporosis, Paget's disease, osteomalacia, hyperostotic disease, osteopetrosis and the like. Osteoporosis has particularly a high incidence among over approximately half of the population of women after the menopause and older persons; therefore, diagnosis and effective therapy have been strongly required.
Bone metabolic diseases are accompanied with any effects of the metabolism that are specific to bone at the cellular level in bone. It may be a very useful tool for elucidating the abnormal metabolism to discover, isolate and identify the factors capable of specifically participating in bone metabolism. The present inventors have made earnest studies to find out one of the specific factors to such bone metabolism and finally completed the present invention.
In the concrete, the present inventors particularly identified a protein factor specifically produced by a cell line of osteoblast that plays a major role in bone formation. Moreover, the present invention provides a novel essentially bone-specific protein named OSF-6 that was obtained by the said research, and that has strong homology in terms of various previously known transcription control factors at the amino acid level.
The OSF-6 of the present invention is to be produced according to any conventional genetic engineering techniques well-known to those skilled in the art using the DNA sequence as disclosed herein. The OSF-6 or a fragment thereof is to also be produced from the amino acid sequence as disclosed herein according to a chemical peptide synthesis method. Moreover, the partial sequence of the DNA sequence of the present OSF-6 as disclosed herein, which is highly specific to other transcription control factors, is to be synthesized with a 15-50 base length according to a conventional oligonucleotide synthesis method, and it is to be applied as the DNA probe for distinction and diagnosis of bone-derived cells. The distinction of bone-derived cells is useful, especially for distinction of origin of metastatic recurrent cancers, and may thus provide an adequate therapy for recurrent cancers. And further, of the partial peptides of OSF-6, the epitome portion of a peptide capable of recognizing an antibody may be employed for producing a specific monoclonal antibody to OSF-6. The monoclonal antibody thus obtained is useful for the identification of bone-derived cells using an immunological cellular tissue staining method.
The prior findings will be summarized below as reported in regard to the protein of the group of transcription control factors to which the OSF-6 provided by the present invention belongs.
It may be said in higher organisms that the phenomena of cell differentiation or cell development are the results of gene expression intricately regulated, temporally and spatially. Accordingly, it will be important in order to comprehend such life phenomena to elucidate control mechanisms of gene expression. In many cases, gene expression is controlled at the transcription level. It has been elucidated from previous studies on eucaryotes that there are protein transcription control factors which bind specific sequences on DNA and activate the transcription reaction by RNA polymerase. From the fact that homeobox genes essential to morphogenesis like muscle cell differentiation factor MyO D, and some oncogenes, such as jun or fos, encode transcription factors having DNA binding ability, it may be apparent that the transcription control factors could play an important role in proliferation or differentiation of cells, and further in the development of tissue formation at the individual level like their assembly.
It is known by the molecular structures of transcription control factors cloned so far and analysis of the functional domains thereof that typical transcription control factors could have a DNA-binding region and a transcription-activating region, and that they could be classified into some corresponding motives [Mitchel and Tjian, (1989) Science, Vol., 245, pp. 371-378]. The DNA-binding region as classified there mentions a zinc-finger, homeodomain, leucine zipper, and the like. The zinc-finger has a conformation containing Zn.sup.2+ bound via adjacent 2 cysteine residues and 2 histidine residues, and has been found in many DNA-binding proteins. The homeodomain is the region highly conserved in the homeobox gene product, which plays an important role in morphogenesis, and has the helix-turn-helix structure of approximately 60 amino acids. The leucine zipper has the Leu residues at seven intervals in a helix structure, is lined on the same dimension, and may participate in dimer formation. It is believed that the direct interaction with DNA would be made with the adjacent basic amino acid region. On the other hand, the acidic amino acid region, the glutamic acid-rich region, the proline-rich region and the like, have been reported as the transcription-activating regions as classified. The mechanism to promote transcription in these transcription-activating regions has not yet been elucidated; the recent study using yeast has, however, revealed the direct interaction of the transcription-activating region with the transcription-initiating complex. Moreover, it was suggested that the interaction could be done via the non-DNA binding protein factor called the adaptor or the mediator, and the cDNA cloning of the molecule believed to function actually as the adaptor was performed [Berger et al., (1992) Cell, Vol. 70, pp. 251-265)].
2. Description of the Prior Art
Osteoblasts, which play a major role in the process of bone formation, are considered to differentiate from undifferentiated mesenchymal cells. Although it is also known that these cells are able to differentiate into myoblasts and adipocytes in addition to osteoblasts, there are many aspects that remain unclear in the process in which pre-osteoblasts differentiate into mature osteoblasts. BMPs (bone morphogenetic proteins) (Wozney et al., (1988) Science, Vol. 242, pp. 1528, 1534), which are known bone formation factors, are reported to have the ability to induce differentiation of a certain type of stromal cell line into osteoblast-like cells (Yamaguchi et al., (1991) J. Cell Bio., Vol. 113, pp. 681-687, and Thies et al., (1992) Endocrinology, Vol. 130, pp. 1318-1324). However, the mechanism by which the expression of bone-associated genes is brought about still remains almost unknown. In addition, the differential gene expressions of type I collagen, alkaline phosphatase, as well as other bone matrix proteins such as osteopontin and osteocalcin, are observed in the differentiation process of osteoblasts. The regulation of expression of these genes is considered to be important in the differentiation of osteoblasts, or in other words, in the process of bone formation. For example, known transcription control factors involved in bone formation include a vitamin D receptor, which is bound to the upstream region of the osteopontin or osteocalcin gene and promotes transcription thereof, as well as c-Fos, which is expressed in large amounts in bone and cartilaginous tissues in the generation process. However, there are still no reports of transcription control factors specific to osteoblasts.